Since the identification of the CD4 molecule by the W3/25 antibody and the demonstration for this antibody of a biological effect of immunosuppression, the anti-CD4 antibodies have attracted noteworthy clinical interest. They have been targets of extensive attention on the part of the pharmaceutical industry, for which they represent a market of tens of millions of individuals grouping together pathologies as varied as the autoimmune disorders, graft rejection and HIV infection.
There exist at present around one hundred anti-CD4 antibodies, most of which have been reported to have an immunosuppressive activity. However, the majority of these antibodies are only used as immunologic reagents (ELISA, flow cytometry, diagnostics, etc.). Around ten of the antibodies have been developed for therapeutic use but, at present, only five anti-CD4 antibodies are effectively in the process of clinical trials: 1) The OKT4a antibody: of murine origin, the OKT4a antibody recognizes at the surface of the human CD4 molecule an epitope overlapping the CDR2-like region of the D1 domain. 2) CLENOLIXIMAB™: this anti CDR2-like antibody of the D1 domain of CD4 was characterized and studied under the name of KELIXIMAB™ and improved by Newman et al. by mutation to have not only a capacity to bind the Fc receptor diminished by a factor of 1000, but also a half-life improved from four to nine days. 3) The Hu5A8 antibody: this is an IgG4/κ anti-domain D2 of CD4 of murine origin humanized by the method of CDR-grafting (Boon et al., 2002). It is claimed that this antibody has a strong anti-HIV activity (Burkly et al., 1992; Moore et al., 1992; Reimann et al., 1997). Its non-immunosuppressive nature and the lack of harmful effect on the level of circulating T CD4+ lymphocytes (Reimann et al. 1997) make it an excellent candidate in the framework of the treatment of AIDS in combination or in those patients who are refractory to the currently available therapies. 4) The MDX-CD4 antibody: this anti-domain D1 of CD4 is totally human and did not require any engineering (Fishwild et al., 1996). Isolated from a murine hybridoma after immunization of transgenic mice, it is presently used in a form re-expressed in CHO cells which has no effect on its specificity, its affinity for the CD4 molecule nor its functional characteristics (Fishwild et al., 1999). 5) The CAMPATH-9H antibody which is an IgG1/κ humanized by the CDR-grafting method based on a rat antibody (Gorman et al., 1991). Obtained by immunization of rats, this anti-domain D1 antibody of CD4 was initially described as improving the quality of the treatment of an autoimmune disorder by another antibody, CAMPATH-1H itself directed against the CD52 molecule. A new engineering approach was able to produce this antibody in myeloma cells of non-secretor NSO mice and not induce the mechanism of complement dependent cytotoxicity (CDC) and only slightly the mechanisms of antibody dependent cellular cytotoxicity (ADCC) (Peakman et al., 1994).
Lastly, the 13B8.2 monoclonal antibody (IOT4a) was described at the end of the 1980's as an antiviral agent preventing the proliferation of HIV. These data immediately led to Phase I clinical testing in a trial involving seven AIDS patients (Dhiver et al., 1989). Many other clinical trials were carried out with this 13B8.2 antibody (Schedel et al., 1993; Deckert et al., 1996; Schedel et al., 1999). All of these studies confirmed the clinical benefit of the administration of the antibody on the progression of the disease. The studies carried out by the group of C. Devaux resulted in the determination of the mechanism of action of the 13B8.2 antibody (for review, Briant and Devaux, 2000).
There was demonstrated the existence of an association between its antiviral activity and an inhibition of the proliferation of HIV resulting from the inactivation of the cascades of intracellular signalizations normally enabling the induction of the expression of the viral genome.
That antiviral activity could be the consequence of many factors such as the induction of a negative signal not dependent on p56lck nor on the HIV co-receptors or the inhibition of a functional mechanism implicating the dimerization oligomerization of the CD4 molecule. In both cases, the 13B8.2 antibody inhibits the signalization cascades implicating the pathway of the MAPkinases, pathways that normally lead to the nuclear translocation of the NF-κB transcription factor.
The 13B8.2 monoclonal antibody was described as directed against the CDR3-like loop of the D1 domain of the CD4 molecule (Sattentau et al., 1989; Corbeau et al., 1993; Houlgatte et al., 1994).
The first clinical trials undertaken with the 13B8.2 antibody were predictive of the therapeutic potential of that molecule. Nevertheless, its development into a product of pharmaceutical interest encountered numerous problems inherent in the structure and the murine origin of the antibody. In fact, the first clinical trials involving AIDS patients and incorporating the 13B8.2 antibody in its original murine version revealed the induction of a HAMA response which, although relatively mild, still interfered with the therapeutic efficacy of the molecule (Dhiver et al., 1989; Deckert et al., 1996; Schedel et al., 1999).
In order to limit the immunogenicity of that molecule and to develop a product of therapeutic interest, a recombinant chimeric Fab fragment of the 13B8.2 antibody was developed and prepared after isolation and sequencing of the domains V of the 13B8.2 antibody (Chardès et al., 1999) by expression by the baculovirus/insect cell system for presenting the domains V of the heavy and light chains of the parental murine antibody fused respectively with the human CH1-γ1 and Cκ domains.
It has been shown that the recombinant chimeric Fab fragment of the 13B8.2 anti-CD4 antibody is capable of binding CD4 with the same epitopic specificity as the parental antibody. Furthermore, this fragment reproduces the biological properties of the 13B8.2 antibody from (1) an antiviral point of view, inhibition of the activation of the HIV promoter and reverse-transcriptase activity and (2) from an immunosuppression point of view, inhibition of the cellular activation subsequent to the presentation of antigens and mixed lymphocyte reactions.
That functionality has already been demonstrated for the chimeric Fab fragment of the antibody (Benkirane et al., 1995). Chimerization thus does not affect its capacity to inhibit, viral proliferation even if a relatively lesser efficacy is noted.
The evaluation of the biological properties of the recombinant Fab fragment shows that this Fab fragment exhibits an activity similar to that of the parental antibody both in terms of inhibition of the activation of the HIV promoter and in the capacity to inhibit the secretion of IL-2 of a T CD4+ lymphocyte in response to the presentation of antigen. (The chimeric mouse-human anti-CD4 Fab 13B8.2 expressed in baculovirus inhibits both antigen presentation and HIV-1 promoter activation. (Bès C. et al. Human Antibodies 10 (2001) 67-76).
The recombinant chimeric Fab fragment of the 13B8.2 antibody possesses the double advantage of being of smaller size than a complete antibody, which improves its pharmacodynamics constants and enables it in a sense to escape the immune system, but also to exhibit good immunosuppressive qualities, which would explain the absence of HAMA type response noted during the preliminary clinical phases with the complete antibody in its murine form.